Aflatoxin B1 (AFB1) is a potent liver carcinogen and also a potent inhibitor of liver nucleolar RNA synthesis. The specific aims of this proposal are: 1, to firmly establish that the binding of AFB1 after activation is preferential to the active nucleolar chromatin, 2, to separate and identify the p-3 chromatin components that bind AFB1 in vivo and in vitro and to quantitatively correlate the binding with the degree inhibition of nucleolar RNA synthesis, and 3, to determine whether the critical target site of AFB1 binding is the chromosomal protein or the nucleolar DNA that is truly responsible for the inhibition of nucleolar RNA synthesis. To accomplish specific aim 1, we propose: 1) to use DNase I digestion of 3H-AFB1 bound nucleolar chromatin, 2) to directly compare the specific activities of 3H-AFB1 in total nucleolar DNA and in rDNA after 3H-AFB1 binding; and 3) to directly compare the specific activities of 3H-AFB1 in the total nucleolar chromatin and in the active nucleolar chromatic (i.e. p-3 Fraction) after 3H-AFB1 binding. To accomplish specific aim 2, we propose: 1) to separate and identify the nucleolar p-3 chromosomal components that bind AFB1. This includes: a) to isolate and identify histones from p-3 franction that bind AFB1, b)to isolate and identify the high mobility group proteins from p-3 fraction that bind AFB1, and c) to isolate the total chromosomal proteins from p-3 fraction by high salt extraction and to identify the AFB1 binding proteins by hydroxyapatite column chromatography, 2) to quantitatively correlate the AFB1 binding in vivo and in vitro at several dose levels with the degree of inhbition of RNA synthesis. This includes: a) to quantitatively correlate AFB1 binding to p-3 DNA with the degree inhibition of RNA synthesis, and b) to quantitavely correlate AFB1 binding to p-3 histones, high mobility group proteins and other nonhistone proteins with the degree inhibition of RNA synthesis. To accomplish specific aim 3, it is planned: 1) to isolate rDNAs from control and AFB1 treated groups and test the template efficiency with RNA polymerase I, and 2) to isolate histone, high mobility group proteins and other non-histones from both control and AFB1 treated p-3 chromatin and test the chromatin template efficiency will the reconstituted "mini chromatin", or "hybrid chromatin" containing normal p-3 DNA. It is believed that these proposed experiments will provide insights into the underlying molecular mechanisms of AFB1 inhibition of rat liver nuclear RNA synthesis.